Embryo implantation, the process by which the blastocyst attaches and implants in the uterus, leads to the establishment of an intimate relationship between the embryo and the endometrium. Implantation is one of the most important limiting factors in establishing a successful pregnancy. It is a complex process involving active interactions between the blastocyst and the uterus. The uterus must undergo dramatic morphological and physiological changes to transform itself from a non-receptive to a receptive state. This differentiation process is largely mediated by the coordinated effects of the ovarian hormones, which act through their intracellular receptors to regulate gene expression, and hence to influence cellular proliferation and differentiation. It is also regulated by the blastocyst.
While the details of the exact molecular events occurring in the uterus during this differentiation process towards receptivity are still unknown, in principle it can be predicted that a unique set of genes is up- or down-regulated in a temporally and spatially specific manner. Indeed, induction of specific genes in the uterus during the peri-implantation period, including those encoding some growth factors and cytokines, has been reported. However, given the complexity and the as-yet imprecisely defined molecular mechanism of the process, many other molecules critical for implantation are still unidentified.
In their earlier application U.S. Ser. No. 10/485,313 (which is herein incorporated in its entirety by reference) the inventors used the mouse as a model in a search for molecules important in the early stage of implantation. In the mouse on day 4.5 of pregnancy (vaginal plug=day 0), the uterus undergoes dramatic morphological changes in association with cell proliferation and differentiation, leading to the acquisition of a receptive state. This uterine remodeling is associated with an increase in vascular permeability at implantation sites. The inventors hypothesized that the proliferation and differentiation of endometrial cells at this time is associated with up- or down-regulation of a number of genes.
To identify uterine genes which are potentially critical for uterine receptivity, the inventors used the technique of RNA differential display (DDPCR) and compared the mRNA expression patterns of implantation and interimplantation sites on day 4.5 of pregnancy (Nie G, Li Y, Batten L, Griffiths B, Wang J, Findlay J & Salamonsen L A (2000) Uterine expression of alternatively spliced mRNAs of mouse splicing factor SC35 during early pregnancy. Mol. Hum. Reprod. 6: 1131-1139). One of the mRNA molecules identified as being differently regulated between the two sites was found to encode a protein molecule, with a predicted serine protease motif. This protein was significantly homologous to SEQ ID NO: 3 described in WO 00/39149 (Barnes), and had significant homology to HtrA type proteins. These proteins were not previously suggested to be involved in embryo implantation.
Accordingly, further work was performed to identify the role of this protein in pregnancy and to identify potential uses. The cDNA encoding this protein was isolated, and its uterine expression during early pregnancy in the mouse examined; the protein is up-regulated in the pregnant mouse uterus from day 4.5 and further increased in the implantation site (including the maternal deciduum and the fetus and the placenta) from day 8.5 onwards. The observed expression pattern indicated a role for this protein in implantation, placentation and early pregnancy.
It is an aim of a preferred embodiment of the present invention to further study the role of serine proteases in fertility and to develop tools for such study. It is also an aim of a preferred embodiment of the invention to provide a test for fertility related disorders.